The invention relates to methods for synthesizing L-fucose and L-fucose analogs. More particularly, the invention is directed to enzymatic methods for synthesizing L-fucose and L-fucose analogs and to the L-fucose analogs synthesized thereby.
L-fucose 1 is a naturally occurring sugar widely found in Nature. It is found in many bacterial and plant glycosides and polysaccharides (Lindberg et. al., MTP Int. Rev. Ser. One, Carbohydr. 1973, 7, 319; Jann, X et. al. In I. W. Shutherland (Ed.), Surface Carbohydrates of the Prokaryotic Cell, Academic Press, New York, 1977, pp 247-287; Aspinall, G. O. et. al. MTP Int. Rev. Sci., Ser. One, Carbobydr. 1973, 7, 285). L-fucose is 25 sometimes found sulfated (Percival, E et. al. Methods Carbohydr. Chem. 1962, 1, 195; Larsen, B et. al., Acta Chem. Scand. 1966, 20, 219; Chandrasekaran, E. V. et. al. Biochemistry 1995, 34, 2925). It has also been found in oligosaccharides of human milk (Kobata, A. In M. I. Horowitz, W. Pigman (Eds.), The Glycoconjugates, Vol. 1, Academic Press, New York, 1977, pp 423-440). In addition, L-fucose is found in many glycolipids (Hakomori, S.-I. Prog. Biochem. Pharmacol. 1975, 10, 167; Mckibbin, J. M. J. Lipid Res. 1978, 19, 131) and glycoproteins including several families of blood-group antigens (Loyd, K. O. MTP Int. Rev. Sci., Ser. Two, Carbohydr. 1976, 7, 251; Kornfeld et. al. Annu. Rev. Biochem. 1976, 45, 217). It is found in cell-surface oligosaccharides, such as the tetrasaccharide sialyl Lewis x (Lex) on neutrophils, as part of selectin ligands involved in cell adhesion (Phillips, M. et. al. Science 1990, 250, 1130; Waltz, G. et. al., Science 1990, 250, 1132; Lowe, J et. al. Cell 1990, 63, 475) and cancer metastasis processes (Paulson, J. C. In The Receptors; Conn, M., Ed.; Academic Press: New York, 1985; Vol. 2, pp 131-219; Paulson, J. C. In Adhesion: its role in inflammatory disease; Harlan, J.; Liu, D., Eds.; W. H. Freeman: New York, 1992; Chapter 2, p 19; Springer, T. et. al. Nature 1991, 349, 196; Lasky, L. Science 1992, 258, 964; Rice, G. et. al. Science 1989, 246, 1303; Bevilacqua, M P et. al. Proc. Natl. Acad. Sci. U.S.A. 1987, 84, 9238.
Given the importance of sialyl Lex and derivatives as potential therapeutic agents for the treatment of inflammatory diseases, the development of an efficient synthesis of 1 has been a subject of interest in glycotechnology (Wong et. al. J. Am. Chem. Soc. 1992, 114, 9283; Wong, et. al. J. Am. Chem. Soc. 1993, 115, 7549; Wong, et. al. J. Am. Chem. Soc. 1995, 117, 66; Mulligan, M. S. et. al. Nature 1993, 364, 149; Flowers, H. M. Adv. Carbohydr. Chem. Biochem. 1981, 39, 279).
Organic synthesis may be employed to produce L-fucose 1. Tanimura discloses an organic synthesis of L-fucose 1 in 9 steps with a 1% overall yield, starting from L-arabinose (Tanimura, A. et. al. Eisei Shikenjo Hokoku 1959, 77, 123; Chem. Abstr. 1961, 55, 12306). An improved organic synthesis of L-fucose 1 is disclosed by Dejter-Juszyzynski using D-galactose as a starting material and providing a 15% overall yield (4.88 mmol) in 4 steps (Dejter-Juszynski, M. et. al. Carbohydr. Res. 1973, 28, 144). A synthesis for the preparation of L-fucose from D-glucose has produced 19.3% of L-fucose and afforded 1.69 mmol of L-fucose in 5 steps (Chiba, T., Chem. Pharm. Bull. 1979, 27, 2838). L-fucose has also been synthesized using D-mannose in 54.8% yield and 6 steps (Gesson, J. et. al. Tetrahedron Lett. 1992, 33, 3637) or using methyl xcex1-D-mannopyranoside in 24% overall yield affording 0.26 mmol of L-fucose in 8 steps (Wong, C.-C. Carbohydr. Res. 1981, 95, 131). In general, the known organic syntheses of L-fucose 1 employ multichemical transformations and result in low yields.
There is no known method for synthesizing L-fucose 1 enzymatically.
Commercially, L-fucose 1 is obtained from natural sources. The preferred natural source is Fucoidan, i.e. a substance extracted from kelp (Schweiger, R. G. (To Kelco Co.), U. S. Pat. No. 3,240,775 Mar. 15, 1966, Appl. Jul. 30, 1962; Chem. Abstr. 1966, 65, 2342).
The synthesis of L-fuculose-1-phosphate 4 has been previously carried out from dihydroxyacetone phosphate 2 and L-lactaldehyde 3 via recombinant L-fuculose aldolase-catalyzed aldolic condensation. In addition, the synthesis of L-fuculose-1-phosphate 4 has been performed via L-fucose isomerase-catalyzed isomerization of L-fucose 1, coupled with L-rhamnulose kinase (Fessner et. al. Tetrahedron: Asymmetry 1993, 4, 1183; Fessner et. al. Angew. Chem. Int. Ed. Engl. 1991, 30, 555; Fessner et. al. Tetrahedron Lett. 1992, 33, 5231; Wong, C.-H. et. al. J. Org. Chem. 1991, 56, 6280). D/L lactaldehyde dimethylacetal 6 has been synthesized and reported by Wong et. al. J. Am. Chem. Soc. 1986, 108, 7812.
L-fuculose 5 has been prepared from L-fucose 1 using a cell free extract of an E. coli mutant strain in the presence of a borate buffer (Green, M.; Cohen, S. S. J. Biol. Chem. 1956, 219, 557) via bacterial oxidation of L-fucitol by Aerobacter suboxidans (Williams, D. T.; Jones, J. K. N. Can. J. Chem. 1967, 45, 741) and from 4 via enzyme-catalyzed phosphate hydrolysis (Fessner et. al. Tetrahedron: Asymmetry 1993, 4, 1183; Wong, C.-H. et. al. J. Org. Chem. 1991, 56, 6280).
L-fuculose-1-phosphate aldolase is prepared from recombinant E.Coli cells using the methodology as described in Wong, C.-H. et. al. J. Am. Chem. Soc. 1994, 116, 6191 and Wong, C.-H. Bioorg. Med. Chem. 1995, 3, 945. E.coli which express recombinant aldolase may be obtained from American Type Culture Collection (ATCC number 86984). E.coli which express recombinant isomerase may be obtained from American Type Culture Collection (ATCC number 87024). Acid phosphatase is commercially available and can be purchased from Sigma chemical company.
What is needed is a simple method for enzymatically synthesizing L-fucose 1 and L-fucose analogs for providing good yields at low cost.
One aspect of the invention is directed to a method for enzymatically synthesizing L-fucose and L-fucose analogs. The method for enzymatically synthesizing L-fucose includes three steps, viz. providing L-fuculose-1-phosphate, enzymatically converting the L-fuculose-1-phosphate to L-fuculose; and then enzymatically converting the L-fuculose to L-fucose.
In a preferred mode, the L-fuculose-1-phosphate is obtained by means of an aldol addition reaction between dihydroxyacetone phosphate and DL-lactaldehyde catalyzed by aldolase or more particularly by L-fucolose-1-phosphate aldolase. The DL-lactaldehyde may be obtained by conversion from DL-lactaldehyde dimethylacetal. In one mode of the invention, the L-fuculose-1-phosphate is purified after its production by the aldol addition reaction and prior to its conversion to L-fuculose. In an alternative mode of the invention, the L-fuculose-1-phosphate is not purified after the aldol addition reaction, i.e., it is employed without purification in a dephosphorylation reaction which converts the L-fuculose-1-phosphate to L-fuculose. In this later instance, the aldol condensation reaction and the dephosphorylation reaction may be performed in a single reaction vessel.
In a preferred mode the dephosphorylation of L-fuculose-1-phosphate to form L-fuculose is catalyzed by acid phosphatase (E.C. 3.1.3.2). The isomerization of the L-fuculose to form L-fucose may then be catalyzed by L-fucose isomerase. In one mode of the invention, the L-fuculose is purified prior to its isomerization to L-fucose. In an alternative mode of the invention, the L-fuculose is not purified, i.e., it is employed without purification in the isomerization reaction which converts the L-fuculose to L-fucose. In this later instance, the dephosphorylation reaction and the isomerization reactions may be performed in a single reaction vessel.
A second aspect of the invention is directed to a method for enzymatically synthesizing L-fucose analogs represented by the following structure: 
wherein R is a substituent selected from the group consisting of H, xe2x80x94CH2, Et, xe2x80x94CH2N3, and xe2x80x94CH2OMe. The method for enzymatically synthesizing L-fucose analogs includes three steps, viz. an enzymatically catalyzed aldol addition reaction for converting a substrate to a first intermediate, an enzymatically catalyzed dephosphorylation for converting a first intermediate to a second intermediate, and an enzymatically catalyzed isomerization for converting the second intermediate to the L-fucose analog.
In a preferred mode of this second aspect of the invention, an aldolase is employed for catalyzing the aldol addition reaction for converting a substrate to a first intermediate. A preferred substrate is a compound represented by the following structure: 
wherein R is a substituent selected from the group consisting of H, xe2x80x94CH2, Et, xe2x80x94CH2N3, and xe2x80x94CH2OMe. A preferred aldolase is L-fuculose-1-phosphate aldolase. The product of the aldol addition reaction is the first intermediate, represented by the following structure: 
wherein R is a substituent selected from the group consisting of H, xe2x80x94CH2, Et, xe2x80x94CH2N3, and xe2x80x94CH2OMe.
The first intermediate is then converted to a second intermediate in a reaction catalyzed by acid phosphatase (E.C. 3.1.3.2). The second intermediate is represented by the following structure: 
wherein R is a substituent selected from the group consisting of H, xe2x80x94CH2, Et, xe2x80x94CH2N3, and xe2x80x94CH2OMe. The second intermediate is then converted to the L-fucose analog in a reaction catalyzed by L-fucose isomerase.
In one mode of the invention, the first intermediate is purified after its production by an aldol addition reaction and prior to its conversion to the second intermediate. In an alternative mode of the invention, the first intermediate is not purified after the aldol addition reaction, i.e., it is employed without purification in a dephosphorylation reaction which converts the first intermediate to the second intermediate. In this instance, the condensation reaction and the dephosphorylation reaction may be performed in a single reaction vessel.
In a preferred mode the dephosphorylation of the first intermediate to form the second intermediate is catalyzed by catalyzed by acid phosphatase (E.C. 3.1.3.2). The isomerization of the second intermediate to form the L-fucose analog may then be catalyzed by L-fucose isomerase. In one mode of the invention, the second intermediate is purified prior to its isomerization to form the L-fucose analog. In an alternative mode of the invention, the second intermediate is not purified, i.e., it is employed without purification in the isomerization reaction to form the L-fucose analog. In this later instance, the dephosphorylation reaction and the isomerization reactions may be performed in a single reaction vessel.
A third aspect of the invention is directed to a method for enzymatically synthesizing L-fucose analogs represented by the following structure: 
wherein R is Me. The method for enzymatically synthesizing L-fucose analogs includes three steps, viz. an enzymatically catalyzed aldol addition reaction for converting a substrate to a first intermediate, an enzymatically catalyzed dephosphorylation for converting a first intermediate to a second intermediate, and an enzymatically catalyzed isomerization for converting the second intermediate to the L-fucose analog.
In a preferred mode of this third aspect of the invention, an aldolase is employed for catalyzing the aldol addition reaction for converting a substrate to a first intermediate. A preferred substrate is a compound represented by the following structure: 
wherein R is Me. A preferred aldolase is L-fuculose-1-phosphate aldolase. The product of the aldol addition reaction is the first intermediate, represented by the following structure: 
wherein R is Me.
The first intermediate is then converted to a second intermediate in a reaction catalyzed by acid phosphatase (E.C. 3.1.3.2). The second intermediate is represented by the following structure: 
wherein R is Me. The second intermediate is then converted to the L-fucose analog in a reaction catalyzed by L-fucose isomerase.
In one mode of the invention, the first intermediate is purified after its production by means of an aldol addition reaction and prior to its conversion to the second intermediate. In an alternative mode of the invention, the first intermediate is not purified after the aldol addition reaction, i.e., it is employed without purification in a dephosphorylation reaction which converts the first intermediate to the second intermediate. In this instance, the aldol addition reaction and the dephosphorylation reaction may be performed in a single reaction vessel.
In a preferred mode the dephosphorylation of the first intermediate to form the second intermediate is catalyzed by catalyzed by acid phosphatase (E.C. 3.1.3.2). The isomerization of the second intermediate to form the L-fucose analog may then be catalyzed by L-fucose isomerase. In one mode of the invention, the second intermediate is purified prior to its isomerization to form the L-fucose analog. In an alternative mode of the invention, the second intermediate is not purified, i.e., it is employed without purification in the isomerization reaction to form the L-fucose analog. In this later instance, the dephosphorylation reaction and the isomerization reactions may be performed in a single reaction vessel.
A fourth aspect of the invention is directed to the compound synthesized by the method of the third aspect of the invention and represented by the following structure: 
wherein R is Me.